Pulmonary neutrophilic inflammation and chronic infection with Pseudomonas aeruginosa (P. aeruginosa) are the major features of cystic fibrosis (CF). The resulting obstructive lung disease is the primary cause for the morbidity and mortality in CF patients. Our long term goal is to gain insight into the pathogenesis of recurrent infection and inflammation to identify novel targets for CF therapy. The focus of this study is on the role of high mobility group box 1 (HMGB1) in P. aeruginosa infection and inflammation in CF. Extracellular HMGB1 is a newly identified mediator for inflammation in acute lung injury and sepsis. Preliminary studies in an in vitro system indicate that airway HMGB1 plays a critical role in neutrophilic inflammation and in modulating the host's ability to clear P. aeruginosa in CF. In Specific Aim 1 of this application, a CF mouse model of P. aeruginosa infection will be used to test the hypotheses that HMGB1 in CF airway evokes pulmonary inflammation and suppresses P. aeruginosa clearance. Both specific anti-HMGB1 antibodies and ethyl pyruvate, a pharmacological agent which can effectively reduce airway HMGB1 and is safe in humans, will be used in these experiments. Experiments designed in Specific Aim 2 will determine the role of airway epithelia in the elevated levels of airway HMGB1 in CF and the underlying mechanisms for the release of HMGB1 from CF epithelia. Western blot analyses will be used to determine the HMGB1 release in conditioned culture media of CF airway epithelia. The mechanisms of HMGB1 release from CF epithelia will be examined using pharmacological inhibitors of acetylase and CF transgenic epithelia. Specific Aim 3 will focus on testing the hypotheses that HMGB1 suppresses bacterial clearance by: (a) decreasing the ability of macrophages to migrate and phagocytose P. aeruginosa via altering actin cytoskeleton organization, and (b) reducing bacterial killing by decreasing nitric oxide (NO) production. Actin cytoskeleton organization and NO production will be examined via histochemical and colorimetric Greiss analyses. Completion of this project will help us to understand the detailed mechanisms through which HMGB1 mediates the pathogenesis of CF. Inhibition of HMGB1 by ethyl pyruvate, which is in a phase II clinical trail, may provide a novel clinical tool for the treatment of CF patients.